Functionalizing Yeast Lipid Droplets as Versatile Biomaterials.

Lipid droplets (LD) are dynamic cellular organelles of ≈1 µm diameter in yeast where a neutral lipid core is surrounded by a phospholipid monolayer and attendant proteins. Beyond the storage of lipids, opportunities for LD engineering remain underdeveloped but they show excellent potential as new biomaterials. In this research, LD from yeast Saccharomyces cerevisiae is engineered to display mCherry fluorescent protein, Halotag ligand binding protein, plasma membrane binding v-SNARE protein, and carbonic anhydrase enzyme via linkage to oleosin, an LD anchoring protein. Each protein-oleosin fusion is coded via a single gene construct. The expressed fusion proteins are specifically displayed on LD and their functions can be assessed within cells by fluorescence confocal microscopy, TEM, and as isolated materials via AFM, flow cytometry, spectrophotometry, and by enzyme activity assay. LD isolated from the cell are shown to be robust and stabilize proteins anchored into them. These engineered LD function as reporters, bind specific ligands, guide LD and their attendant proteins into union with the plasma membrane, and catalyze reactions. Here, engineered LD functions are extended well beyond traditional lipid storage toward new material applications aided by a versatile oleosin platform anchored into LD and displaying linked proteins.

Beneath the surface: Evolution of methane activity in the bacterial multicomponent monooxygenases.

The bacterial multicomponent monooxygenase (BMM) family has evolved to oxidise a wide array of hydrocarbon substrates of importance to environmental emissions and biotechnology: foremost amongst these is methane, which requires among the most powerful oxidant in biology to activate. To understand how the BMM evolved methane oxidation activity, we investigated the changes in the enzyme family at different levels: operonic, phylogenetic analysis of the catalytic hydroxylase, subunit or folding factor presence, and sequence-function analysis across the entirety of the BMM phylogeny. Our results show that the BMM evolution of new activities was enabled by incremental increases in oxidative power of the active site, and these occur in multiple branches of the hydroxylase phylogenetic tree. While the hydroxylase primary sequence changes that resulted in increased oxidative power of the enzyme appear to be minor, the principle evolutionary advances enabling methane activity occurred in the other components of the BMM complex and in the recruitment of stability proteins. We propose that enzyme assembly and stabilization factors have independently-evolved multiple times in the BMM family to support enzymes that oxidise increasingly difficult substrates. Herein, we show an important example of evolution of catalytic function where modifications to the active site and substrate accessibility, which are the usual focus of enzyme evolution, are overshadowed by broader scale changes to structural stabilization and non-catalytic unit development. Retracing macroscale changes during enzyme evolution, as demonstrated here, should find ready application to other enzyme systems and in protein design.

Horizontal gene transfer of three co-inherited methane monooxygenase systems gave rise to methanotrophy in the Proteobacteria.

The critical role that bacterial methanotrophs have in regulating the environmental concentrations of the potent greenhouse gas, methane, under aerobic conditions is dependent on monooxygenase enzymes which oxidise the substrate as both a carbon and energy source. Despite the importance of these organisms, the evolutionary origins of aerobic methane oxidation capability and its relationship to proteobacterial evolution is not well understood. Here we investigated the phylogenetic relationship of proteobacterial methanotrophs with related, non-methanotrophic bacteria using 16S rRNA and the evolution of two forms of methane monooxygenase: membrane bound (pMMO and pXMO) and cytoplasmic (sMMO). Through analysis we have concluded that extant proteobacterial methanotrophs evolved from up to five ancestral species, and that all three methane monooxygenase systems, pMMO, pXMO and sMMO, were likely present in the ancestral species (although pXMO and sMMO are not present in most of the present day methanotrophs). Here we propose that the three monooxygenase systems entered the ancestral species by horizontal gene transfer, with these likely to have pre-existing physiological and metabolic attributes that supported conversion to methanotrophy. Further, we suggest that prior to these enzyme systems developing methane oxidation capabilities, the membrane-bound and cytoplasmic monooxygenases were already both functionally and phylogenetically associated. These results not only suggest that sMMO and pXMO have a far greater role in methanotrophic evolution than previously understood but also implies that the co-inheritance of membrane bound and cytoplasmic monooxygenases have roles additional to that of supporting methanotrophy.

Functional assessment of plant and microalgal lipid pathway genes in yeast to enhance microbial industrial oil production.

As promising alternatives to fossil-derived oils, microbial lipids are important as industrial feedstocks for biofuels and oleochemicals. Our broad aim is to increase lipid content in oleaginous yeast through expression of lipid accumulation genes and use Saccharomyces cerevisiae to functionally assess genes obtained from oil-producing plants and microalgae. Lipid accumulation genes DGAT (diacylglycerol acyltransferase), PDAT (phospholipid: diacylglycerol acyltransferase), and ROD1 (phosphatidylcholine: diacylglycerol choline-phosphotransferase) were separately expressed in yeast and lipid production measured by fluorescence, solvent extraction, thin layer chromatography, and gas chromatography (GC) of fatty acid methyl esters. Expression of DGAT1 from Arabidopsis thaliana effectively increased total fatty acids by 1.81-fold above control, and ROD1 led to increased unsaturated fatty acid content of yeast lipid. The functional assessment approach enabled the fast selection of candidate genes for metabolic engineering of yeast for production of lipid feedstocks.

Metabolic engineering of lipid pathways in Saccharomyces cerevisiae and staged bioprocess for enhanced lipid production and cellular physiology.

Microbially produced lipids have attracted attention for their environmental benefits and commercial value. We have combined lipid pathway engineering in Saccharomyces cerevisiae yeast with bioprocess design to improve productivity and explore barriers to enhanced lipid production. Initially, individual gene expression was tested for impact on yeast growth and lipid production. Then, two base strains were prepared for enhanced lipid accumulation and stabilization steps by combining DGAT1, ΔTgl3 with or without Atclo1, which increased lipid content ~ 1.8-fold but reduced cell viability. Next, fatty acid (FA) biosynthesis genes Ald6-SEACSL641P alone or with ACC1** were co-expressed in base strains, which significantly improved lipid content (8.0% DCW, 2.6-fold than control), but severely reduced yeast growth and cell viability. Finally, a designed two-stage process convincingly ameliorated the negative effects, resulting in normal cell growth, very high lipid productivity (307 mg/L, 4.6-fold above control) and improved cell viability.

Self-Assembled Enzyme Nanoparticles for Carbon Dioxide Capture.

Enzyme-based processes have shown promise as a sustainable alternative to amine-based processes for carbon dioxide capture. In this work, we have engineered carbonic anhydrase nanoparticles that retain 98% of hydratase activity in comparison to their free counterparts. Carbonic anhydrase was fused with a self-assembling peptide that facilitates the noncovalent assembly of the particle and together were recombinantly expressed from a single gene construct in Escherichia coli. The purified enzymes, when subjected to a reduced pH, form 50-200 nm nanoparticles. The CO2 capture capability of enzyme nanoparticles was demonstrated at ambient (22 ± 2 °C) and higher (50 °C) temperatures, under which the nanoparticles maintain their assembled state. The carrier-free enzymatic nanoparticles demonstrated here offer a new approach to stabilize and reuse enzymes in a simple and cost-effective manner.

Biochemical characterization of a halotolerant feruloyl esterase from Actinomyces spp.: refolding and activity following thermal deactivation.

Ferulic acid esterases (FAE, EC. 3.1.1.73) hydrolyse the linkage between hemicellulose and lignin and thus have potential for use in mild enzymatic pretreatment of biomass as an alternative to thermochemical approaches. Here, we report the characterization of a novel FAE (ActOFaeI) obtained from the bacterium, Actinomyces sp. oral which was recombinantly expressed in Escherichia coli BL21 in two forms: with and without its putative signal peptide. The truncated form was found to have <10 % relative activity compared to the full length and was more prone to aggregation after purification. The enzyme with retained peptide demonstrated 2 to 4-fold higher activity against methyl caffeate and methyl p-coumarate, with specific activities of 477.6 and 174.4 U mg(-1) respectively, than the equivalent activities of the benchmark FAE from Aspergillus niger A and B. ActOFaeI retained activity over a broad pH range with a maximum at 9 but >90 % relative activity at pH 6.5 and an optimum reaction temperature of 30 °C. ActOFaeI increased activity by 15% in high salt conditions (1000 mMNaCl) and its thermal unfolding temperature improved from 41.5 °C in standard buffer to 74 °C in the presence of 2500 mM sodium malonate. ActOFaeI also released ferulic acid from destarched wheat bran when combined with a xylanase preparation. After treatment above the thermal denaturation temperature followed by cooling to room temperature, ActOFaeI demonstrated spontaneous refolding into an active state. ActOFaeI displays many useful characteristics for enzymatic pretreatment of lignocellulose and contributes to our understanding of this important family.

Diversity of Δ12 fatty acid desaturases in santalaceae and their role in production of seed oil acetylenic fatty acids.

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.

Cross-feeding promotes heterogeneity within yeast cell populations.

Cellular heterogeneity in cell populations of isogenic origin is driven by intrinsic factors such as stochastic gene expression, as well as external factors like nutrient availability and interactions with neighbouring cells. Heterogeneity promotes population fitness and thus has important implications in antimicrobial and anticancer treatments, where stress tolerance plays a significant role. Here, we study plasmid retention dynamics within a population of plasmid-complemented ura3∆0 yeast cells, and show that the exchange of complementary metabolites between plasmid-carrying prototrophs and plasmid-free auxotrophs allows the latter to survive and proliferate in selective environments. This process also affects plasmid copy number in plasmid-carrying prototrophs, further promoting cellular functional heterogeneity. Finally, we show that targeted genetic engineering can be used to suppress cross-feeding and reduce the frequency of plasmid-free auxotrophs, or to exploit it for intentional population diversification and division of labour in co-culture systems.

Exploring engineering strategies that enhance de novo production of exotic cyclopropane fatty acids in Saccharomyces cerevisiae.

Cycloalkanes have broad applications as specialty fuels, lubricants, and pharmaceuticals but are not currently available from renewable sources, whereas, production of microbial cycloalkanes such as cyclopropane fatty acids (CFA) has bottlenecks. Here, a systematic investigation was undertaken into the biosynthesis of CFA in Saccharomyces cerevisiae heterologously expressing bacterial CFA synthase. The enzyme catalyzes formation of a 3-membered ring in unsaturated fatty acids. Monounsaturated fatty acids in phospholipids (PL) are the site of CFA synthesis; precursor cis-Δ9 C16 and C18 fatty acids were enhanced through OLE1 and SAM2 overexpression which enhanced CFA in PL. CFA turnover from PL to storage in triacylglycerols (TAG) was achieved by phospholipase PBL2 overexpression and acyl-CoA synthase to increase flux to TAG. Consequently, CFA storage as TAG reached 12 mg g-1 DCW, improved 3-fold over the base strain and >22% of TAG was CFA. Our research improves understanding of cycloalkane biosynthesis in yeast and offers insights into processing of other exotic fatty acids.

Coassembled Multicomponent Protein Nanoparticles Elicit Enhanced Antibacterial Activity.

The waning pipeline of the useful antibacterial arsenal has necessitated the urgent development of more effective antibacterial strategies with distinct mechanisms to rival the continuing emergence of resistant pathogens, particularly Gram-negative bacteria, due to their explicit drug-impermeable, two-membrane-sandwiched cell wall envelope. Herein, we have developed multicomponent coassembled nanoparticles with strong bactericidal activity and simultaneous bacterial cell envelope targeting using a peptide coassembly strategy. Compared to the single-component self-assembled nanoparticle counterparts or cocktail mixtures of these at a similar concentration, coassembled multicomponent nanoparticles showed higher bacterial killing efficiency against Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli by several orders of magnitude (about 100-1,000,000-fold increase). Comprehensive confocal and electron microscopy suggest that the superior antibacterial activity of the coassembled nanoparticles proceeds via multiple complementary mechanisms of action, including membrane destabilization, disruption, and cell wall hydrolysis, actions that were not observed with the single nanoparticle counterparts. To understand the fundamental working mechanisms behind the improved performance of coassembled nanoparticles, we utilized a "dilution effect" system where the antibacterial components are intermolecularly mixed and coassembled with a non-antibacterial protein in the nanoparticles. We suggest that coassembled nanoparticles mediate enhanced bacterial killing activity by attributes such as optimized local concentration, high avidity, cooperativity, and synergy. The nanoparticles showed no cytotoxic or hemolytic activity against tested eukaryotic cells and erythrocytes. Collectively, these findings reveal potential strategies for disrupting the impermeable barrier that Gram-negative pathogens leverage to restrict antibacterial access and may serve as a platform technology for potential nano-antibacterial design to strengthen the declining antibiotic arsenal.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Modulating the hydrophobicity of cellulose by lipase-catalyzed transesterification.

The production of hydrophobic and oil resistant cellulosic fibers usually requires severe chemical treatments and generates toxic by-products. Alternative approaches such as biocatalysis use milder conditions; lipase-catalyzed methods for grafting nanocellulose with hydrophobic ester moieties have been reported. Here, we investigate the lipase-catalyzed esterification of cellulose fibers, in native form or pretreated with 1,4-ß-glucanases, and cellulose nanocrystals (CNC) in solvent-free conditions. The fibers were compared for degree of ester formation after incubation with methyl myristate and lipase at 50 °C. After washing, the grafting of fatty esters on cellulose was confirmed by ATR-FTIR and the degree of substitution determined by 13C CP/MAS NMR (from 0.04 up to DS 0.1) confirming successful esterification. Optical photothermal infrared (O-PTIR) spectroscopy showed strongly localized presence of ester moieties on cellulose. Functional properties mirrored the degree of substitution of the cellulose materials whereby cellulose esters made with glucanase-pretreatment produced the highest water contact angle of 117° ± 9 and esterified cellulose blended at 10 % w/w content in paper composites showed significant differences in hydrophobicity and lipophilicity compared to plain paper. The esterification of cellulose was completely reversed by lipase treatment in aqueous media. These ester-functionalized fibers show potential in a wide range of packaging applications.

Rapid production of multimeric RNA aptamers stabilized by a designed pseudo-circular structure in E. coli.

RNA aptamers bind specifically and selectively to various macromolecules, cell surfaces, and viruses and find broad applications as biosensors, diagnostics, and in therapeutic treatments and drug delivery. Currently, RNA aptamer production is via in vitro methods. Herein, a new E. coli-based approach has been demonstrated for the rapid production of multimeric RNA aptamer transcripts that are protected from degradation by burying the 5' and 3' ends of the transcript in a designed double-stranded spacer. Multimeric and fluorescent RNA aptamers were produced stably in vivo and readily isolated from RNase III-deficient cells, and their full functionalities were shown by binding assays and fluorescence measurements. This approach shows promise as a rapid and scalable bioprocess for the production of RNA aptamers at low cost.

An Ultrastable Self-Assembled Antibacterial Nanospears Made of Protein.

Protein-based nanomaterials have broad applications in the biomedical and bionanotechnological sectors owing to their outstanding properties such as high biocompatibility and biodegradability, structural stability, sophisticated functional versatility, and being environmentally benign. They have gained considerable attention in drug delivery, cancer therapeutics, vaccines, immunotherapies, biosensing, and biocatalysis. However, so far, in the battle against the increasing reports of antibiotic resistance and emerging drug-resistant bacteria, unique nanostructures of this kind are lacking, hindering their potential next-generation antibacterial agents. Here, the discovery of a class of supramolecular nanostructures with well-defined shapes, geometries, or architectures (termed "protein nanospears") based on engineered proteins, exhibiting exceptional broad-spectrum antibacterial activities, is reported. The protein nanospears are engineered via spontaneous cleavage-dependent or precisely tunable self-assembly routes using mild metal salt-ions (Mg2+ , Ca2+ , Na+ ) as a molecular trigger. The nanospears' dimensions collectively range from entire nano- to micrometer scale. The protein nanospears display exceptional thermal and chemical stability yet rapidly disassemble upon exposure to high concentrations of chaotropes (>1 mm sodium dodecyl sulfate (SDS)). Using a combination of biological assays and electron microscopy imaging, it is revealed that the nanospears spontaneously induce rapid and irreparable damage to bacterial morphology via a unique action mechanism provided by their nanostructure and enzymatic action, a feat inaccessible to traditional antibiotics. These protein-based nanospears show promise as a potent tool to combat the growing threats of resistant bacteria, inspiring a new way to engineer other antibacterial protein nanomaterials with diverse structural and dimensional architectures and functional properties.

Effect of seed coat microstructure and lipid composition on the hydration behavior and kinetics of two red bean (Phaseolus vulgaris L.) varieties.

Bean hydration is a crucial, yet complex process affected both by inherent bean properties (intrinsic) and properties of the soak medium (extrinsic) whose relationship with hydration kinetics are not clear. This work investigated the effect of temperature (30-80°C) on the hydration behavior and kinetics of two commonly consumed Phaseolus vulgaris L. beans-red kidney and small red beans. Both beans have similar morphology (except size), chemical composition and seed coat surface structure but, surprisingly, their hydration behaviors differed markedly. Small red hydration followed a sigmoidal shape (to 70°C) with a prominent lag phase at 30-40°C, while fast hydrating red kidney beans followed a downward concave shape, suggesting different mechanisms of water uptake. The hydration behaviors for red kidney and small red beans were modeled using Peleg (R2 ≥0.97) and sigmoidal (R2 ≥0.99) models, respectively. Water uptake rate increased, while equilibrium moisture content decreased for both bean varieties with increasing temperature. A simplified model was developed for small red beans which can predict hydration as a function of temperature and time with good accuracy ( M mod = M expt . × 1.06 - 0.56 ${M_{{\rm{mod}}}} = {M_{{\rm{expt}}{\rm{.}}}}\ \times 1.06 - 0.56$ , R2  = 0.99, RMSE = 4.5). Microscopic examination of cross sections of seed coats showed nearly double parenchyma layer thickness in small red bean and seed coat lipid analysis showed significantly higher proportion of saturated fatty acids which, together, may be responsible for slower water uptake observed in small red beans. PRACTICAL APPLICATION: Hydration is the crucial stage in dry bean processing, which precedes other processes like cooking, extraction, fermentation, sprouting, and consumption. Industrial bean processing is still slow and batch process and needs improvement. This work shows the effect of various bean properties and soaking medium temperature on the hydration behavior of two commonly consumed red beans. The work also modeled the hydration kinetics of two Phaseolus vulgaris L. beans which can be adopted by bean processors for process design and optimization.

Fatty acid distribution and polymorphism in solid lipid particles of milkfat and long chain omega-3 fatty acids.

Saturated fatty acid-containing lipids, such as milkfat, may protect long chain polyunsaturated fatty acids in fish oil when blended together into solid lipid particles (SLPs). One of the main challenges of SLPs is structural polymorphism, which can lead to expulsion of the protected component during prolonged storage. To investigate this phenomenon, the change in thermal and crystalline behaviours, and fatty acid distribution, were analysed in SLPs of fish oil and milkfat during storage at different temperatures for up to 28 days. X-ray diffraction analysis showed changes in molten and crystalline states occurred even at -22 °C. Room temperature (21 °C) storage led to more than 45% molten state but SLPs retained their initial shape. Confocal Raman Spectroscopy of the SLPs showed the distribution of fatty acids was not uniform, with 10 µm outermost layer of predominantly saturated fatty acids likely responsible for the intact SLP shape and stability of the core.

Engineering Self-Assembled Endolysin Nanoparticles against Antibiotic-Resistant Bacteria.

Antibiotic resistance represents a serious global health concern and has stimulated the development of antimicrobial nanomaterials to combat resistant bacteria. Protein-based nanoparticles combining characteristics of both proteins and nanoparticles offer advantages including high biocompatibility, attractive biodegradability, enhanced bioavailability and functional versatility. They have played an increasing role as promising candidates for broad applications ranging from biocatalysts and drug delivery to vaccine development to cancer therapeutics. However, their application as antibacterial biomaterials to address challenging antibiotic-resistance problems has not been explicitly pursued. Herein, we describe engineering protein-only nanoparticles against resistant Gram-positive bacteria. A self-assembling peptide (P114) enables the assembly of a phage lytic enzyme (P128) into nanoparticles in response to pH reduction. Compared to native P128 and monomeric P114-P128, P128 nanoparticles (P128NANO) demonstrated a stronger bactericidal ability with high potency at lower concentrations (2-3-fold lower), particularly for methicillin-resistant Staphylococcus aureus strains. In addition, P128NANO showed an enhanced thermal (up to 65 °C) and storage stability and elicited extensive damages to bacterial cell walls. These remarkable antibacterial abilities are likely due to the P128NANO nanostructure, mediating multivalent interactions with bacterial cell walls at increased local concentrations of endolysin. The engineered endolysin nanoparticles offer a promising antimicrobial alternative to conventional antibiotics.

Harnessing disorder: onychophorans use highly unstructured proteins, not silks, for prey capture.

Onychophora are ancient, carnivorous soft-bodied invertebrates which capture their prey in slime that originates from dedicated glands located on either side of the head. While the biochemical composition of the slime is known, its unusual nature and the mechanism of ensnaring thread formation have remained elusive. We have examined gene expression in the slime gland from an Australian onychophoran, Euperipatoides rowelli, and matched expressed sequence tags to separated proteins from the slime. The analysis revealed three categories of protein present: unique high-molecular-weight proline-rich proteins, and smaller concentrations of lectins and small peptides, the latter two likely to act as protease inhibitors and antimicrobial agents. The predominant proline-rich proteins (200 kDa+) are composed of tandem repeated motifs and distinguished by an unusually high proline and charged residue content. Unlike the highly structured proteins such as silks used for prey capture by spiders and insects, these proteins lack ordered secondary structure over their entire length. We propose that on expulsion of slime from the gland onto prey, evaporative water loss triggers a glass transition change in the protein solution, resulting in adhesive and enmeshing thread formation, assisted by cross-linking of complementary charged and hydrophobic regions of the protein. Euperipatoides rowelli has developed an entirely new method of capturing prey by harnessing disordered proteins rather than structured, silk-like proteins.